To provide protection from pathogens without concomitant autoimmunity, the immune system must retain functional T cells specific for peptides derived from microbial antigens and must silence T cells specific for self peptides. Maintaining self-tolerance is particular problematic for developmentally-regulated self antigens that appear after the secondary lymphoid organs are populated with "response-ready" mature T cells. Earlier work suggests that peripheral T cells make the decision to respond or not based on the context in which the antigen is presented. For example, antigen-presentation in the presence of inflammation generally results in immunity whereas antigen-presentation without inflammation results in tolerance. However, the cellular and molecular mechanisms that underlie peripheral tolerance have not been clearly established. We propose to address this by studying in detail a novel system in which the in vivo fate of a small population of adoptively-transferred TCR transgenic T cells specific for an ovalbumin peptide can be directly monitored by flow cytometry or immunohistology. Using this system we plan to identify the mechanism(s) that underlie a classical means of inducing peripheral tolerance, i.e., in vivo administration of soluble antigen in the absence of an adjuvant. Specifically, we plan to determine whether the unresponsive antigen-specific T cells that remain after in vivo injection of antigen in the absence of an adjuvant have defects in lymphokine production and B cell helper function, and if so, determine if these defects are long-lasting. It will be determined whether T cell unresponsiveness is induced because: (a) antigen is presented to antigen-specific T cells in the absence of CD28 costimulation, or the presence of negative signals mediated by CTLA-4 or NFAT-1; (b) Th2-like suppressor cells inhibit Th1 cytokine production by the antigen-specific T cells; or (c) antigen-specific T cells do not differentiate into effector lymphokine producing T cells because inflammatory cytokines are not present at the time of initial antigen presentation. Finally, it will be determined whether unresponsiveness is maintained by defects in the TCR or CD28 signal transduction pathways and the signaling molecules that are affected will be identified. The advantage of our approach is that by directly tracking the fate and functional capabilities of antigen-specific T cells within mixtures of other lymphoid cells, we hope to be able to reduce the complexity of the in vivo situation to a point where mechanisms can be clearly revealed.